All-trans-retinoic acid (RA) induces differentiation of acute promyelocytic leukemia (APL) cells both in vitro and in vivo and is an alternative to cytotoxic chemotherapy in the treatment of APL. However, despite a complete remission rate of about 90%, most patients relapse and are resistant to further treatment with RA. This resistance primarily is due to an increased systemic catabolism of RA. We examined the catabolism of RA by the human acute promyelocytic leukemia cell line NB4 and the human myeloid leukemia cell line HL60. NB4 cells converted RA to 4-hydroxy-, 4-oxo-retinoic acid and a more polar unidentified retinoid at a much greater rate than HL60 cells. The catabolism of RA was induced by pretreatment of the cells with RA. We found that 4-hydroxyphenylretinamide (4-HPR) inhibited the catabolism of RA. This inhibition was dose-dependent and greater, on a molar basis, than the inhibition seen with the cytochrome P450 inhibitor, ketoconazole, or two synthetic retinoids, Ch55 and Am80. 4-HPR alone was a poor inducer of differentiation of NB4 cells. However, in combination with RA it markedly enhanced RA-induced differentiation and increased the level of retinoylation, the covalent binding of RA to proteins. This is the first report of a specific enzymatic reaction involving RA that is inhibited by 4-HPR. These results suggest that some retinoid analogs, including 4-HPR, may have clinical utility because of their ability to increase the biological half-life of RA.